Viral Immunogenic Compositions

ABSTRACT

Disclosed herein are immunogenic compositions for producing immediate and sustained immunity to infectious viral and bacteriological pathogens. A univalent immunogenic composition is disclosed comprising an isolated antigen and a polynucleotide formulated into a nanoparticle or liposome. Furthermore, multivalent immunogenic compositions are disclosed comprising multiple univalent immunogenic compositions. Also disclosed, are methods of inducing protective or therapeutic immune responses in individuals comprising administering one or more univalent immunogenic compositions.

CROSS- REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/514,613 filed Aug. 3, 2011, the entirecontents of which is incorporated by reference herein.

FIELD OF THE INVENTION

The present application is drawn to immunogenic compositions forpreventing and treating Influenza virus infection, and methods of makingand using the immunogenic compositions.

BACKGROUND OF THE INVENTION

Every year many people catch “the flu” and for most it is aself-limiting illness with recovery occurring between two to threeweeks. Influenza is however, a potentially more severe infectiousdisease which is highly contagious. It is estimated that there arebetween three to five million cases of severe flu worldwide resulting inup to 500,000 deaths annually. There are two types of influenza virusesthat cause epidemic human disease: the Influenza A virus which has manydifferent strains, and the Influenza B virus which consists of a singlestrain. Current flu vaccines are a mixture of viral antigens preparedfrom selected strains of influenza A virus and from the single strain ofthe influenza B virus. The current vaccination programs are aimed atcontaining the seasonal flu. Health authorities from many countriescollaborate with one another to predict the strain of influenza A virusthat is likely to cause flu in the upcoming months. For the most part,these types of vaccination programs have worked well to contain theseasonal flu, although they become less effective when the flu epidemicis caused by a different influenza A strain than was predicted. This isbecause vaccination against one influenza A strain affords little if anyimmunity against a different influenza A strain. Once the strain isidentified it takes about six months to manufacture large quantities ofthe appropriate vaccine. As such, one of the drawbacks of thesevaccination programs is the time it takes to formulate a vaccine whichmay not even provide any protection against the influenza A straincausing seasonal flu in a particular season. For instance, if it takessix months to formulate a vaccine which provides little or no immunitybecause the predicted strain is not the one causing the infection, itwill take another six months to develop the appropriate vaccine at whichpoint there may be a threat for a major flu pandemic.

It would therefore be desirable to develop an effective immunogeniccomposition that is tailored to any flu strain that may arise and thatcan be produced in large quantities and deployed rapidly. Further, itwould be desirable if the immunogenic composition could elicit animmediate and prolonged immunity to the flu virus.

The desirability of developing such an immunogenic composition becomeseven more urgent when one considers that every ten to twenty years a flustrain arises that infects a large proportion of the world's populationand has the potential to kill tens of millions of people. For example,within the last century there have been three major flu pandemics: theSpanish Flu in 1918-1920 which killed 40 million to 100 million people;the Asian Flu in 1957-1958 which killed 1 million to 1.5 million people;and the Hong Kong Flu in 1968-1969 which killed about 1 million people.

Another major obstacle in developing an effective vaccination program isthat the influenza A virus is constantly changing. For example, it isknown that the influenza A virus may undergo successive point mutationsduring replication (i.e. genetic drift) and can gradually acquire thecapacity over time to become more virulent. Another way in which newstrains of flu virus appear is when different flu strains may co-infecta host and exchange genetic information with each other (i.e. geneticshift) resulting in the appearance of a new flu strain with differentinfective properties and pathogenic potential from either of its parentstrains.

Different strains of the influenza A virus are known to infect a varietyof animal species, including birds and pigs, and some of these strainsmay cross specie lines and infect humans. If the infected human alsohappens to be co-infected with another strain of influenza virus thereis a possibility that the two viral strains may exchange geneticinformation resulting in the appearance of a new strain. For example,there is great concern that the avian flu virus found in domestic andwild birds may infect a human who is carrying a human flu virus strainand exchange genetic information to form a new flu viral strain with thecapacity to propagate from human to human resulting in a worldwidepandemic.

There are intensive efforts being made to develop antiviral drugs andvaccines to combat the threat of a worldwide pandemic. Much of theresearch on flu vaccine is based on traditional methods of growing thevirus in embryonated eggs or in tissue cultured cells and thenextracting the viral proteins to prepare a vaccine. This has workedfairly well for seasonal flu vaccines where health agencies are able toidentify new strains of flu virus as they arise and have sufficient timeto prepare the appropriate vaccine. However, the vaccination programstill has to be repeated each year as immunity to one strain of the fluvirus will not fully protect against any new strain that develops.

Another major obstacle with the traditional vaccination program is thatbecause of mass transit and the large numbers of air travelers, any flustrain that arises in one country can quickly spread among othercountries and infect the general population. Moreover, because of themany potential permutations of influenza virus strains that can arise,it is difficult to prepare a vaccine in a timely fashion using thetraditional method of growing the virus in embryonated eggs or culturingviral infected cells. Thus, the time frame for identifying new viralstrains and preparing large amounts of the correct vaccine has grownincreasingly short which continues to be one of the major hindrancesfacing the traditional vaccination program. There are also verystringent manufacturing safety protocols that must be employedthroughout production when a pathogenic virus is used to prepare avaccine. Even the more recent methods of using genetic engineering toprepare recombinant viral antigens still require several months toprepare a suitable vaccine that may not be deployed in time to beeffective.

The current vaccination programs that were developed for the seasonalflu are unable to produce the large quantities of vaccine in the limitedtime available to protect against a flu pandemic. Even the most currentvaccine production methods using cell culture requires about 4 months toproduce the appropriate vaccine (Novartis 2008). This time frame isclearly inadequate when the response time required to prevent a flupandemic is measured in days.

The difficulty is further compounded because the current vaccines areonly designed to elicit a classical immune response which can takeseveral weeks to develop after vaccination. Moreover, to develop fullimmunity two doses of vaccine are often required. For example, The WorldHealth Organization reported in 2007 that “It is unlikely that apotential pandemic could be successfully contained through the use ofvaccination alone. Vaccination, with some exceptions, is not normallyused to “contain” outbreaks of seasonal influenza. Full immunity islikely to require two doses of vaccine, and to take 3 weeks to developafter the first vaccination. Moreover, the degree of antigenic matchbetween stockpiled vaccine and a potential H5N1 influenza pandemic viruscannot be known. There will also be considerable logistical challengesin vaccinating large numbers (possibly millions) of people over arelatively short period of time.”

In view of the deficiencies of the current vaccination programs it wouldbe desirable to develop an immunogenic composition that could provideimmediate and long-term immunity to any flu strain that should arise. Itwould also be desirable to be able to produce large quantities of theappropriate flu immunogenic composition very quickly so that it can bedeployed in time to be effective.

SUMMARY OF THE INVENTION

This application discloses a method of preparing a multivalentimmunogenic composition against the influenza A virus.

Disclosed herein, is a univalent immunogenic composition comprising anisolated antigen and a polynucleotide formulated into a nanoparticle orliposome.

In one embodiment, the univalent immunogenic composition furthercomprises an adjuvant. In another embodiment, the adjuvant is alum. Inanother embodiment, the adjuvant is formulated into the nanoparticle orliposome or admixed with the nanoparticle or liposome formulatedunivalent immunogenic composition.

In another embodiment, the isolated antigen is an influenza virusantigen such as a hemagglutinin, a neuraminidase, or a matrix protein.

In one embodiment, the polynucleotide is a synthetic polynucleotidecomprising double stranded DNA or double stranded RNA. In anotherembodiment, the double stranded DNA or double stranded RNA comprisesnucleotide repeats. In yet another embodiment, the polynucleotide ispoly-IC or poly-ICLC.

In another embodiment the liposome is a self-assembling liposome system.In yet another embodiment, the liposome further comprises monophosphoryllipid A or pullulans.

Also disclosed herein, is a method of inducing a protective ortherapeutic immune response in an individual comprising administeringone or more univalent immunogenic compositions, wherein a firstunivalent immunogenic composition comprises a first antigen. In anotherembodiment, a second univalent immunogenic composition comprises asecond antigen. In another embodiment, a third univalent immunogeniccomposition comprises a third antigen.

In another embodiment, the one or more univalent immunogeniccompositions are administered by a method selected from the groupconsisting of scarification, intradermal injection, intramuscularinjection, sub-lingual administration, and inhalation. In otherembodiments, the one or more univalent immunogenic compositions areadministered at different times or substantially simultaneously. Inanother embodiment, the one or more univalent immunogenic compositionsare each administered by injection to the individual at the same site onthe individual or at different sites on the individual. In anotherembodiment, the one or more univalent immunogenic compositions arecombined prior to injection into a multivalent immunogenic composition.

Also disclosed herein, is a multivalent immunogenic compositioncomprising two or more univalent immunogenic compositions, wherein eachof the univalent immunogenic compositions comprises a different antigen.

In yet another embodiment, the approach of stockpiling all thecomponents required to prepare a complete univalent or multivalentimmunogenic composition or the univalent immunogenic compositionsthemselves is disclosed. The components are viral antigen andpolynucleotide packaged into a nanoparticle or liposome. Once anepidemic flu virus is identified, the stockpiled immunogenic compositioncomponents, or univalent immunogenic compositions, can be quicklyassembled into the appropriate univalent immunogenic compositionsdirected against the identified viral antigens. The selected univalentimmunogenic compositions are then administered separately or they may becombined to form a multivalent immunogenic composition beforeadministration.

The same general principles enumerated herein can be applied topreparing immunogenic compositions against other viral and bacterialpathogens. It can be particularly advantageous when applied tovaccination programs against potential pandemics and the threat ofbioterrorism.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a comprehensive integrated approach to developing aneffective vaccination program that can be employed to protect againstinfluenza. The same general principles can also be employed to prepareimmunogenic compositions against other viral and microbiologicalpathogens. As used herein, an “immunogenic composition” refers to acomposition capable of inducing a protective or therapeutic immuneresponse, with or without an adjuvant, and includes, in part, an antigenagainst which the protective or therapeutic immune response is elicited.An immunogenic composition is an example of an immunogenic composition.

First, a number of basic univalent immunogenic compositions are preparedwith each immunogenic composition directed against a single antigenselected from the group of hemagglutinin (H), neraminidase (N), andmatrix (M) antigens of the influenza A virus. An individual's immuneresponse is enhanced by presenting only one purified antigen at a timeto an antigen processing and/or immunocompetent cell. Rapid andpersistent immunity is achieved by incorporating a double-strandedpolynucleotide chain with each specific viral antigen. The combinedantigen and polynucleotide moieties are incorporated into a nanoparticleor liposomal univalent immunogenic composition formulation.Administration of the immunogenic composition stimulates an immediateprotective interferon response followed by the classical immune responseto the antigen. To shorten the response time to the threat of anepidemic, the individual components of the univalent immunogeniccomposition i.e. viral antigen, polynucleotide, and nanoparticle orliposome supplies are separately stockpiled and stored. Once the suspectflu virus strain is identified the appropriate immunogenic compositioncomponents are then assembled into separate univalent immunogeniccompositions. Individuals may receive one or more univalent immunogeniccompositions directed against different flu antigens to obtain broadimmunity. Several different univalent immunogenic compositions may alsobe combined to produce a multivalent immunogenic composition forconvenience in administration. Vaccination may be by dermal, sub-dermal,or intramuscular injection, or by inhalation, or sub-lingualadministration.

Further disclosed herein is a method of immunogenic compositionpreparation that is appropriate for flu immunogenic compositions. Theimmunogenic composition is designed to confer immediate and long-termprotection against viral infection and is particularly useful againstpandemic flu viruses. It is also designed to be an effective immunogeniccomposition by using prestocked components that can be easily formulatedinto a immunogenic composition and quickly deployed where needed.

The Influenza A Virus

The influenza A virus consists of a viral RNA core surrounded by a viralcoat composed of multiple units of hemagglutinin (H) protein andmultiple units of neraminidase (N) protein. There are 16 known varietiesof H protein and 9 known varieties of the N protein. These aredesignated as H1 thru H16, and N1 thru N9 respectively. Each strain offlu virus has one H component and one N component making up its viralcoat. For example, one avian flu virus is composed of H5N1 one mildstrain of human flu was found to be H9N and a virulent strain of humanflu was found to be H1N1.

When different strains of flu virus co-infect the same host they mayexchange genetic information resulting in a reshuffling of the H and Ncomponents to form a new viral strain. For example, if the flu strainH5N1 infects a human who is also infected with a flu virus such as H1N2,it is possible that a new flu strain H5N2 or a new flu strain H1N1 couldbe created. The new strain will exhibit a different infectivity andvirulence profile from its parent strains.

Based on the number of different H antigens and N antigens exhibited bythe known influenza A viruses, the total number of permutations thatcould theoretically result from genetic reshuffling is calculated bymultiplying the 16 H varieties by the 9 N varieties to give 144 possibleflu strains. In addition there are minor antigenic variations (i.e.clades) that will result in even greater antigenic diversity.

This application discloses that it would be more advantageous tostockpile individual components of the immunogenic composition and tothen assemble the final immunogenic composition once the flu strain wasidentified rather than trying to develop a complete immunogeniccomposition against a predicted flu strain. This approach eliminates thetime required to grow the predicted virus strain and produce largequantities of the immunogenic composition. It also provides a moreefficient method of ensuring that the immunogenic composition isspecific for the virus. For example, many companies are stockpilingvaccines prepared against the avian flu virus H5N1 because of thepossibility that the H5N1 virus strain could become pandemic. But ifthere was a genetic shift and the pandemic flu virus turned out to be aH1N1 strain instead, the H5N1 immunogenic composition would not provideimmunity against the H1N1 virus. However, if there were stockpiles ofthe H1 antigen and the N1 antigen as disclosed herein, then amultivalent immunogenic composition composed of a H1 univalentimmunogenic composition and N1 univalent immunogenic composition wouldbe available and expected to provide better protection as they would betargeted against the appropriate strains causing the virus. Further, thestockpiled immunogenic composition components can be assembled veryquickly and be deployed in an effective time to where they are needed.

It should also be noted that there are antigenic variations within eachtype of isolated influenza A strain. For example, the H5 antigen of theH5N1 virus will show antigenic differences between different H5N1isolates. In developing an immunogenic composition those H5 antigenicsubtypes are selected which show the best cross-reactivity with other H5isolates and/or develop several immunogenic compositions directedagainst selected antigenic sub-types. This also applies for otherinfluenza A strains that exhibit antigen variation within the strain.

In another embodiment of this disclosure, the immunogenic compositionalso includes a purified antigen prepared from the conserved matrix (M1and M2) regions of the flu virus coat.

In order to obtain the best immune response the viral antigens areutilized in a purified and isolated form and each viral antigen ispresented separately to the antigen processing and/or immunocompetentcells. In this manner each clone of immunocompetent cells is programmedto react to only a particular antigen.

In order to obtain optimum immunity against a particular flu strainseveral of these univalent immunogenic compositions directed againstdifferent antigens of that strain are used to vaccinate the individual.For convenience and efficiency, the selected univalent immunogeniccompositions are optionally combined to form a multivalent immunogeniccomposition prior to vaccination.

Viral Antigen

In order to prepare a viral antigen, the different H, N and M viralantigens are purified from viral cultures. In one embodiment, theantigens are prepared from cultured viruses. First, a selected strain ofinfluenza A virus is used to inoculate embryonated eggs or cellcultures. After a period of multiplication the virus is extracted fromthe culture medium and purified using standard production techniquessuch as chromatography. After inactivation, the viral antigens areseparated from other viral components using standard purificationmethods. The method of culturing the virus and the preparation ofpurified viral protein is known to those skilled in the art. The methodsof antigen purification such as gel-filtration, affinity chromatographyand other methods are known to those skilled in the art.

In another embodiment, the purified antigens are prepared by geneticengineering techniques. The genetic code of the particular viral antigenis incorporated into the genomic DNA of a transfected host cell. And thetransfected cell is induced to express the corresponding H or N or Mviral protein. Non-limiting examples of host cells suitable forproduction of the viral antigens disclosed herein include bacteria,yeast, insects, mammalian, or plant cells. For example, if the H1 viralantigen was selected, then the genetic code for H1 would be prepared andused to transform a host cell and the H1 antigen is produced as arecombinant protein. The expression level of the recombinant protein isincreased by the use of suitable promoter genes. The recombinant antigenis purified by downstream processing and prepared for long term storageby lyophilization, or freezing or other similar processes. The method ofgenetic engineering and downstream processing are known to those skilledin the art.

In one embodiment, the antigens are prepared using the Baculovirusvector expression system in insect cells or larvae of the silkwormBombyx mori. In this system the genetic code for the viral antigen islinked to the polyhedron promoter gene and used to produce a transformedbaculovirus. When the transformed baculovirus is used to infect Bombixmori cells they are induced to express the recombinant viral antigen.Similarly, silkworm larvae infected with the transformed baculovirus areinduced to produce large amounts of viral antigen in their hemolymph.This method of baculovirus expression of recombinant proteins anddownstream processing is known to those skilled in the art. The use ofsilkworms as inexpensive “bioreactors” to produce large quantities ofviral antigen is especially suited for developing countries that do nothave access to the advanced and expensive vaccine production capacity ofindustrialized nations.

The viral antigen is isolated and substantially purified such that onlythe desired antigen is present in the composition, excluding undesirableantigens, proteins, lipids, or carbohydrates present in the startingmaterial. For the purposes of the present disclosure, the term antigenincludes full length proteins, and epitopes or fragments of the fulllength proteins which maintain the appropriate immunogenicity or abilityto stimulate an immune response.

Polynucleotide

This application discloses the inclusion of a polynucleotide into theimmunogenic composition. Polynucleotides are molecular chains ofnucleotides usually deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). They can be of cellular or viral origin or they can besynthesized. When administered to an individual the polynucleotide hastwo important functions. First, it has an immune stimulating effect, andsecond it stimulates the production of interferons which are compoundsknown to have an inhibitory effect upon viral infections. For maximumanti-viral effect the polynucleotide is typically double-stranded DNA(dsDNA) or double stranded RNA (dsRNA).

Interferons belong to the large class of glycoproteins known ascytokines. They are natural proteins produced by the cells of the immunesystem in response to challenges by foreign agents such as viruses,parasites and tumor cells. Interferons are produced by a wide variety ofcells in response to the presence of double-stranded RNA, a keyindicator of viral infection. Interferons assist the immune response byinhibiting viral replication within host cells, activating naturalkiller cells and macrophages, increasing antigen presentation tolymphocytes, and inducing the resistance of host cells to viralinfection. When the antigen is presented to matching T and B cells,those cells multiply, attack and degrade the infectious agent. Asdisclosed herein presenting the polynucleotide simultaneously with theantigen to the same antigen processing or immunocompetent cellpotentiates the immune response in addition to inducing the productionof interferons.

In one embodiment of this disclosure a natural dsRNA polynucleotideextracted from any number of known viral or bacterial agents is used.Such agents include influenza A virus, influenza B virus, Sendai virus,E. coli etc. The methods of extraction, amplification using polymerasechain reaction (PCR), and purification of the natural polynucleotide areknown to those of skill in the art. A synthetic polynucleotide can alsobe used. According to this disclosure exemplary syntheticpolynucleotides are double stranded nucleic acids selected from thegroup consisting of: polyinosinic acid and polycytidylic acid (poly-IC),polyadenylic acid and polyuridylic acid (poly-AU), polyinosinic acidanalogue and polycytidylic acid, polyinosinic acid and polycytidylicacid analogue, polyinosinic acid analogue and polycytidylic acidanalogue, polyadenylic acid analogue and polyuridylic acid, polyadenylicacid and polyuridylic acid analogue, and polyadenylic acid analogue andpolyuridylic acid analogue.

The polynucleotide chain may be modified by substituting other basesinto the chain at specified intervals or by attaching additionalcompounds such as poly-L-lysine carboxymethylcellulose to the nucleotidechain. For example poly-IC can be stabilized by adding poly-L-lysine toform a new polynucleotide termed poly-ICLC. Poly-ICLC has shownanti-viral protection against a variety of viruses including vaccinia,hepatitis, influenza, herpesvirus, rabies, Japanese encephalitis, WestNile virus, Ebola virus, and HIV. Of particular note is that intranasalpoly-ICLC administration can protect mice for as long as 3 weeks from anotherwise lethal dose of influenza virus.

In preparing the univalent immunogenic compositions of the presentdisclosure, the polynucleotide is mixed with the viral antigen so thatboth moieties are presented simultaneously to the same antigenprocessing and/or immunocompetent cell. The ratio of antigen topolynucleotide used in the univalent immunogenic composition may rangefrom the order of 100:1 to 1:100. Depending on its potency the totalamount of viral antigen used per univalent immunogenic composition isexpected to be within the range of 1 μg to 100 μg per dose.

In aspects of this embodiment, an immunogenic composition disclosedherein may include a dose of viral antigen that is, e.g., 90-100 μg,80-90 μg, 70-80 μg, 60-70 μg, 50-60 μg, 40-50 μg, 30-40 μg, 20-30 μg,10-20, or less than 10 μg, less than 20 μg, less than 30 μg, less than40 μg, less than 50 μg, less than 60 μg, less than 70 μg, less than 80μg, less than 90 μg, or less than 100 μg. In other embodiments, theratio of antigen to polynucleotide in the univalent immunogeniccomposition is, e.g., 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1,60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1,1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55,1:60, 1:65, 1:70. 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100.

Immunogenic Composition

The immunogenic composition is prepared by combining a viral antigen andpolynucleotide and assembling them into a nanoparticle or liposome. Toprepare the immunogenic composition, the viral antigen andpolynucleotide are bound to the surface of a solid particle matrix suchas colloidal gold particles, chitin nanoparticles, or other similarnanoparticles. The immunogenic composition components may also beincorporated into solid lipid nanoparticles. The methods of preparingnanoparticles include precipitation, homogenization and crystallizationmethods and are known to those skilled in the art.

In another embodiment, the coated nanoparticles are administered inconjunction with an adjuvant to enhance their uptake by antigenprocessing cells. Additionally, the coated nanoparticles may beincorporated into an oil-in-water emulsion which has a known adjuvanteffect. Suitable adjuvants are known to persons of ordinary skill in theart.

The immunogenic composition may also be prepared in the form ofliposomes which are composed of an aqueous core and an outer lipidbilayer membrane. Liposomes are prepared using different lipidcompositions. For example, they are prepared using a mixture of one ormore of the following compounds: phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinsitol,monosialoganglioside, sphingomyelin, distearoylphosphatidylethanolamine,distearoylphosphatidylcholine, dimyristoyl-phosphatidylcholine,dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylcholine and thelike. Cholesterol is also often included to stabilize the bilayer lipidmembrane. The mixed lipids are dissolved in a solvent such as chloroformand dried under vacuum to form a lipid film. The dried lipid film ishydrated in a solution in which the viral antigen and polynucleotide aredissolved. The hydrated lipids will form liposomes entrapping the viralantigen and polynucleotide within the aqueous center of the liposome.Depending on the mixture of lipids used to prepare the liposomes and themethod employed, such as freeze/thawing or homogenization or sonication,the liposomes formed may be multilamella or unilamella, and they mayrange in size from several thousand nanometers in diameter to below onehundred nanometers in diameter. The methods of preparing liposomes areknown to those skilled in the art.

In another embodiment of this disclosure, a lipid formulation mixture isemployed that will spontaneously form liposomes when mixed with anaqueous solution. The methods of manufacturing self-assembling liposomesare known to those skilled in the art.

In yet another embodiment of this disclosure the outer layer ofliposomes may incorporate a known lipid adjuvant such as monophosphoryllipid A (MPL). The basic liposome formulation will consist of aphospholipid compound such as phosphatidylcholine, cholesterol and MPL.

In another embodiment, a self-assembling liposome formulationincorporates an immune stimulating polysaccharide compound such aspullulans. The pullulans compound is anchored into the liposome membranelayer and stimulates uptake by the dendritic cells of the immune system.The dendritic cells are antigen processing cells responsible forinitiating the immune response.

Also within the scope of the present disclosure are kits containing oneor more univalent immunogenic compositions, the components for one ormore immunogenic compositions, and instructions for use.

In one embodiment, the kit contains two, three, four or more isolatedand purified viral antigens, polynucleotides and reagents to preparetwo, three, four or more univalent immunogenic compositions. In someembodiments, the kits also contain an appropriate adjuvant.

In another embodiment, the kit contains two, three, four or moreunivalent immunogenic compositions. In some embodiments, the kits alsocontain an appropriate adjuvant.

Vaccine Administration

The mode of administration of the immunogenic composition may be by aroute including scarification, or by injection, or by inhalation. In oneembodiment, each univalent immunogenic composition is administeredseparately so that the antigen processing cells upon arrival at eachsite of administration will become programmed to develop immunity toonly that particular antigen. In another embodiment, multiple univalentimmunogenic compositions are administered in combination orsimultaneously injected at different locations.

In order to obtain broader immunity to a viral strain it may bedesirable to vaccinate with multiple viral antigens of that strain. Forexample, if the viral strain was avian flu virus with the H5N1 antigenicmakeup, the vaccination program would administer two or more immunogeniccompositions given to the individual. One immunogenic compositionincorporates the recombinant H5 antigen plus a polynucleotide, a secondimmunogenic composition incorporates the recombinant N1 antigen plus thepolynucleotide, while a third immunogenic composition incorporates therecombinant M antigen plus the polynucleotide. The individual injectionsites may be placed close to one another (e.g. using a multineedleinjector) or they may be spaced widely apart (e.g. on different arms);or be given at different times (e.g. on different days). The individualimmunogenic compositions may also be mixed to prepare a multivalentimmunogenic composition and diluted sufficiently so that each antigenprocessing cell is predominantly exposed to only one antigen. The samerationale for preparing tailor-made immunogenic compositions would applyto other influenza strains whose antigenic identity is known. Similarlyfor inhalation or sub-lingual administration, each immunogeniccomposition preparation can be given at different times or on differentdays.

Example of a Vaccine to Treat Pandemics.

For illustrative purposes the preparation of a model immunogeniccomposition to treat an influenza A pandemic is disclosed herein.

The first step is to identify the strains of influenza A virus that posethe greatest threat of causing a pandemic. These will be the viralstrains that are known to infect humans. Specifically, the H, N, and Msubtype of the virus is determined.

The hemagglutinin (H) antigen is responsible for the first phase ofinfection which is the attachment of the virus to the cell; while theneuraminidase (N) antigen is responsible for the last phase of infectionwhich is to cleave and release the viral particle from the infectedcell. Therefore an immunogenic composition prepared against the Hantigen is more effective in preventing viral infection, while aimmunogenic composition prepared against the N antigen is more effectivein limiting the transmission of the virus from host to host.

Based on this reasoning the most important antigens for preparing theimmunogenic composition will be the hemagglutinin antigen sub-types thatshow the highest virulence and capacity for human to human transmission.

To increase host immunity to infection and to limit viral spread,immunogenic compositions against the N antigens from viral strains knownto infect humans are also included.

Again, for illustrative purposes only, the preparation of a modelimmunogenic composition against the avian flu virus using selectedembodiments is disclosed herein. For an avian flu virus with an H5N1antigenic makeup, two univalent immunogenic compositions are prepared.One immunogenic composition is directed against the H5 antigen subtypeand the other immunogenic composition against the N1 antigen. In thisexample, the recombinant H5 and N1 proteins are expressed in silkwormlarvae using the Baculovirus expression system. Briefly, the gene forthe H5 protein is in-frame fused with the polyhedrin (Ph) gene under thecontrol of Ph promoter and used to co-transfect cultured BmN cells withmodified linearized Bombyx mori baculovirus DNA to produce recombinantvirus (rBacPh-H5). The recombinant rBacPh-H5 virus is used to infectsilkworm larvae which are induced to produce large quantities ofrecombinant H5 protein in the hemolymph. Similarly, the gene for the N1protein is in-frame fused with the gene under the control of Ph promoterand used to co-transfect cultured BmN cells with modified linearizedBombyx mori baculovirus DNA to produce recombinant virus (rBacPh-N1).The recombinant rBacPh-N1 virus is used to infect silkworm larvae whichare induced to produce large quantities of recombinant N1 protein in thehemolymph. The recombinant H5 and N1 proteins are purified usingconventional laboratory techniques and stored lyophilized untilrequired.

In this embodiment example, the polynucleotide used in the univalentimmunogenic composition is the synthetic polynucleotide poly-ICLC whichis a potent immunomodulating agent. The methods of preparing syntheticpolynucleotides are known to those skilled in the art. The polypeptidechain can be prepared in bulk and stored in a suitable manner untilrequired.

In this example, a self-assembling liposome incorporating the adjuvantcompound is selected to prepare the univalent immunogenic composition.The liposome components such as phosphatidylcholine, cholesterol andadjuvant are premixed in bulk, vacuum-dried and stored frozen untilrequired. Upon addition of an aqueous solution of the premixed viralantigen and polynucleotide the hydrated lipids will spontaneously formliposomes enclosing the antigen and polynucleotide within. The formedliposomes are lyophilized with a cryoprotectant agent and stored in bulkuntil required.

Other Applications

The concept of stockpiling immunogenic composition components can alsobe applied to seasonal flu where a univalent immunogenic compositionprepared against the influenza B virus can be combined with theunivalent immunogenic compositions prepared against the influenza Avirus strains to give broad protection against developing the flu.

Disclosed herein is an integrated approach to developing an effectiveimmunogenic composition for pandemic influenza and other viruses capableof causing a pandemic. The elements consist of: 1) producing andstockpiling recombinant viral antigens; 2) producing and stockpiling aselected double-stranded polynucleotide; 3) producing and stockpilingmaterials to make nanoparticles or liposomes; 4) assembling therecombinant viral antigen and polynucleotide into a univalentimmunogenic composition; 5) administering different univalentimmunogenic compositions to elicit a sustained immune response; and 6)combining different univalent immunogenic compositions to make amultivalent immunogenic composition to facilitate mass vaccination.

The same general principles enumerated here can be applied to preparingimmunogenic compositions against a variety of other viral and bacterialpathogens. There are many known infectious pathogens that are capable ofcausing pandemics with significant morbidity. The WHO Epidemic andPandemic Alert and Response list includes: anthrax, avian influenza,Crimean-Congo haemorrhagic fever, dengue haemorrhagic fever, Ebolahaemorrhagic fever, hepatitis, influenza, Lassa fever, Marburghaemorrhagic fever, meningococcal disease, plague, rift valley fever,severe acute respiratory syndrome (SARS), smallpox, tularaemia, andyellow fever.

There is also the threat of bioterrorism wherein infectious agents canbe modified to make them more infectious and virulent. The mosteffective means to prepare for these threats is to develop a vaccinationprogram that is effective, safe and can be rapidly deployed.

The above descriptions are given by way of example, and not limitation.Given the above disclosures, one skilled in the art could devisevariations that are within the scope and spirit of the inventiondisclosed herein. Further, the various features of the embodimentsdisclosed herein can be used alone, or in varying combinations with eachother and are not intended to be limited to the specific combinationdescribed herein. Thus, the scope of the claims is not to be limited bythe illustrated embodiments described in this invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A univalent immunogenic composition comprising an isolated antigenand a polynucleotide formulated into a nanoparticle or liposome.
 2. Theunivalent immunogenic composition according to claim 2, furthercomprising an adjuvant.
 3. The univalent immunogenic compositionaccording to claim 2, wherein the adjuvant is alum.
 4. The univalentimmunogenic composition according to claim 1, wherein the adjuvant isformulated into the nanoparticle or liposome or admixed with thenanoparticle or liposome formulated univalent immunogenic composition.5. The univalent immunogenic composition according to claim 1, whereinthe isolated antigen is an influenza virus antigen.
 6. The univalentimmunogenic composition according to claim 5, wherein the influenzavirus antigen is a hemagglutinin, a neuraminidase, or a matrix protein.7. The univalent immunogenic composition according to claim 1, whereinthe polynucleotide is a synthetic polynucleotide comprising doublestranded DNA or double stranded RNA.
 8. The univalent immunogeniccomposition according to claim 7, wherein the double stranded DNA ordouble stranded RNA comprises nucleotide repeats.
 9. The univalentimmunogenic composition according the claim 8, wherein thepolynucleotide is poly-IC or poly-ICLC.
 10. The univalent immunogeniccomposition according to claim 1 wherein the liposome is aself-assembling liposome system.
 11. The univalent immunogeniccomposition according to claim 1, wherein the liposome further comprisesmonophosphoryl lipid A or pullulans.
 12. A method of inducing aprotective or therapeutic immune response in an individual comprisingadministering one or more univalent immunogenic compositions accordingto claim 1, wherein a first univalent immunogenic composition comprisesa first antigen.
 13. The method according to claim 12, wherein a secondunivalent immunogenic composition comprises a second antigen.
 14. Themethod according to claim 12, wherein the one or more univalentimmunogenic compositions are administered by a method selected from thegroup consisting of scarification, intradermal injection, intramuscularinjection, sub-lingual administration, and inhalation.
 15. The methodaccording to claim 12, wherein the one or more univalent immunogeniccompositions are administered by injection to the individual at separatesites on the individual.
 16. The method according to claim 12, whereinthe one or more univalent immunogenic compositions are administered atdifferent times.
 17. The method according to claim 12, wherein the oneor more univalent immunogenic compositions are administeredsubstantially simultaneously.
 18. The method according to claim 12,wherein the one or more univalent immunogenic compositions areadministered by injection to the individual at the same site on theindividual.
 19. The method according to claim 12, wherein the one ormore univalent immunogenic compositions are combined prior to injectioninto a multivalent immunogenic composition.
 20. A multivalentimmunogenic composition comprising two or more univalent immunogeniccompositions according to claim 1, wherein each of the univalentimmunogenic compositions comprises a different antigen.